Cholera toxin (CT) from Vibrio cholerae and heat-labile toxin (LT) from enterotoxigenic Escherichia coli are highly related AB5-type protein toxins that contain a catalytic A1 subunit, an A2 linker, and a cell-binding homopentameric B subunit. CT and LT travel as intact AB5 toxins from the cell surface to the endoplasmic reticulum (ER) of an intoxicated cell. In the ER, the catalytic A1 subunit is dissociated from the rest of the toxin y the action of protein disulfide isomerase (PDI). The free A1 subunit then crosses the ER membrane and enters the cytosol where it elicits a cytopathic effect. CT and LT share ~80% sequence identity across both the A and B subunits. The toxins exhibit high affinity interactions with their shared GM1 ganglioside surface receptor and display similar in vitro ADP-ribosylation activities against their shared Gsa target. Yet CT is much more potent than LT in cell culture: it elicits a greater cytopathic effect than LT, and it does so in a shorter time frame than LT. The difference in toxicity has previously been mapped to the non-catalytic A2 subunit: a chimeric LTA1/CTA2/LTB5 toxin is more potent than wild-type LT and as potent as wild-type CT in vivo, while a chimeric CTA1/LTA2/CTB5 toxin exhibited in vivo toxicity that was less potent than wild-type CT but comparable to wild-type LT. The mechanism by which the A2 subunit influences toxicity remains unknown. We hypothesize the A2 subunit, through its positioning of the A1 subunit within the holotoxin, is responsible for establishing the affinity of PDI-holotoxin binding and the efficiency of A1 displacement from the holotoxin. These events affect the extent of A1 subunit delivery to the cytosol and, thus, intoxication. We have noted the CTA1 and LTA1 subunits are positioned differently within their holotoxins because of the orientation of their respective A2 linkers. Furthermore, we have found the PDI-mediated disassembly of LT is less efficient than the PDI-mediated disassembly of CT. We accordingly predict the limited in vivo potency of LT, in comparison to CT, is due to inefficient disassembly of the LT holotoxin by PDI. This project will use wild-type toxins as well as chimeric CTA1/LTA2/CTB5 and LTA1/CTA2/LTB5 toxins to test our model. These toxins will be used in studies involving (i) surface plasmon resonance (SPR) to calculate the on-rate for PDI-holotoxin binding; (ii) SPR to monitor PDI-mediated holotoxin disassembly in real time; and (iii) cell-based assays to monitor A1 subunit delivery to the cytosol. Toxins containing the LTA2 subunit are predicted to exhibit, in comparison to toxins containing the CTA2 subunit, (i) lower affinity for PDI; (ii) less efficient txin disassembly by PDI; and (iii) reduced levels of the cytosolic A1 subunit. Additional methods in cell biology and biophysics will be used to discount other possible contributions to differential i vivo toxin activity. This project will generate a substantial body of new information on host-LT interactions and will provide a detailed molecular model explaining how the A2 subunit is ultimately responsible for the different in vivo activities of CT and LT.